Nicotinic receptor non-competitive antagonists

ABSTRACT

The present invention relates to compounds that modulate nicotinic receptors as non-competitive antagonists, methods for their synthesis, methods for use, and their pharmaceutical compositions.

FIELD OF THE INVENTION

The present invention relates to compounds that modulate nicotinicreceptors as non-competitive modulators (e.g., non-competitiveantagonists), methods for their synthesis, methods for use, and theirpharmaceutical compositions.

BACKGROUND OF THE INVENTION

Nicotinic receptors are targets for a great number of exogenous andendogenous compounds that allosterically modulate their function. See,Arias, H. R., Binding sites for exogenous and endogenous non-competitiveinhibitors of the nicotinic acetylcholine receptor, Biochimica etBiophysica Acta—Reviews on Biomembranes 1376: 173-220 (1998) and Arias,H. R., Bhumireddy, P., Anesthetics as chemical tools to study thestructure and function of nicotinic acetylcholine receptors, CurrentProtein & Peptide Science 6: 451-472 (2005). The function of nicotinicreceptors can be decreased or blocked by structurally differentcompounds called non-competitive modulators, including non-competitiveantagonists (reviewed by Arias, H. R., Bhumireddy, P., Bouzat, C.,Molecular mechanisms and binding site locations for noncompetitiveantagonists of nicotinic acetylcholine receptors. The InternationalJournal of Biochemistry & Cell Biology 38: 1254-1276 (2006)).

Non-competitive modulators comprise a wide range of structurallydifferent compounds that inhibit receptor function by acting at a siteor sites different from the orthosteric binding site. Receptormodulation has proved to be highly complex. The mechanisms of action andbinding affinities of non-competitive modulators differ among nicotinicreceptor subtypes (Arias et al., 2006). Non-competitive modulators mayact by at least two different mechanisms: an allosteric and/or a stericmechanism.

An allosteric antagonist mechanism involves the binding of anon-competitive antagonist to the receptor and stabilization of anon-conducting conformational state, namely, a resting or desensitizedstate, and/or an increase in the receptor desensitization rate.

In contrast, a straightforward representation of a steric mechanism isthat an antagonist molecule physically blocks the ion channel. Suchantagonists may be termed non-competitive channel modulators (NCMs).Some inhibit the receptors by binding within the pore when the receptoris in the open state, thereby physically blocking ion permeation. Whilesome act only as pure open-channel blockers, others block both open andclosed channels. Such antagonists inhibit ion flux through a mechanismthat does not involve binding at the orthosteric sites.

Barbiturates, dissociative anesthetics, antidepressants, and certainsteroids have been shown to inhibit nicotinic receptors by allostericmechanisms, including open and closed channel blockade. Studies ofbarbiturates support a model whereby binding occurs to both open andclosed states of the receptors, resulting in blockade of the flow ofions. See, Dilger, J. P., Boguslavsky, R., Barann, M., Katz, T., Vidal,A. M., Mechanisms of barbiturate inhibition of acetylcholine receptorchannels, Journal General Physiology 109: 401-414 (1997). Although theinhibitory action of local anesthetics on nerve conduction is primarilymediated by blocking voltage-gated sodium channels, nicotinic receptorsare also targets of local anesthetics. See, Arias, H. R., Role of localanesthetics on both cholinergic and serotonergic ionotropic receptors,Neuroscience and Biobehavioral Reviews 23:817-843 (1999) and Arias, H.R. & Blanton, M. P., Molecular and physicochemical aspects of localanesthetics acting on nicotinic acetylcholine receptor-containingmembranes, Mini Reviews in Medicinal Chemistry 2: 385-410 (2002).

For example, tetracaine binds to the receptor channels preferentially inthe resting state. Dissociative anesthetics inhibit severalneuronal-type nicotinic receptors in clinical concentration ranges, withexamples such as phencyclidine (PCP) (Connolly, J., Boulter, J., &Heinemann, S. F., Alpha 4-beta 2 and other nicotinic acetylcholinereceptor subtypes as targets of psychoactive and addictive drugs,British Journal of Pharmacology 105: 657-666 (1992)), ketamine (Flood,P. & Krasowski M. D., Intravenous anesthetics differentially modulateligand-gated ion channels, Anesthesiology 92: 1418-1425 (2000); and Ho,K. K. & Flood, P., Single amino acid residue in the extracellularportion of transmembrane segment 2 in the nicotinic α7 acetylcholinereceptor modulates sensitivity to ketamine, Anesthesiology 100: 657-662(2004)), and dizocilpine (Krasowski, M. D., & Harrison, N. L., Generalanaesthetic actions on ligand-gated ion channels, Cellular and MolecularLife Sciences 55: 1278-1303 (1999)). Studies indicate that thedissociative anesthetics bind to a single or overlapping sites in theresting ion channel, and suggest that the ketamine/PCP locus partiallyoverlaps the tetracaine binding site in the receptor channel.Dizocilpine, also known as MK-801, is a dissociative anesthetic andanticonvulsant which also acts as a non-competitive antagonist atdifferent nicotinic receptors. Dizocilpine is reported to be anopen-channel blocker of α4β2 neuronal nicotinic receptors. See, Buisson,B., & Bertrand, D., Open-channel blockers at the human α4β2 neuronalnicotinic acetylcholine receptor, Molecular Pharmacology 53: 555-563(1998).

In addition to their well-known actions on monoamine and serotoninreuptake systems, antidepressants have also been shown to modulatenicotinic receptors. Early studies showed that tricyclic antidepressantsact as non-competitive antagonists. See, Gumilar, F., Arias, H. R.,Spitzmaul, G., Bouzat, C., Molecular mechanisms of inhibition ofnicotinic acetylcholine receptors by tricyclic antidepressants.Neuropharmacology 45: 964-76 (2003). Garćia-Colunga et al., report thatfluoxetine, a selective serotonin reuptake inhibitor (SSRI), inhibitsmembrane currents elicited by activation of muscle or neuronal nicotinicreceptors in a non-competitive manner; either by increasing the rate ofdesensitization and/or by inducing channel blockade. See,Garćia-Colunga, J., Awad; J. N., & Miledi, R., Blockage of muscle andneuronal nicotinic acetylcholine receptors by fluoxetine (Prozac),Proceedings of the National Academy of Sciences USA 94: 2041-2044(1997); and Garćia-Colunga, J., Vazquez-Gomez, E., & Miledi. R.,Combined actions of zinc and fluoxetine on nicotinic acetylcholinereceptors, The Pharmacogenomics Journal 4: 388-393 (2004). Mecamylamine,previously approved for the treatment of hypertension, is a classicalnon-competitive nicotinic receptor antagonist, and is also well known toinhibit receptor function by blocking the ion channel. See, Giniatullin,R. A., Sokolova, E. M., Di Angelantonio, S., Skorinkin, A., Talantova,M. V., Nistri, A. Rapid Relief of Block by Mecamylamine of NeuronalNicotinic Acetylcholine Receptors of Rat Chromaffin Cells In Vitro: AnElectrophysiological and Modeling Study. Molecular Pharmacology 58:778-787 (2000).

SUMMARY OF THE INVENTION

The present invention includes compounds of Formula I:

wherein

each of R¹ and R² individually is H, C₁₋₆ alkyl, or aryl-substitutedC₁₋₆ alkyl, or R¹ and R² combine with the nitrogen atom to which theyare attached to form a 3- to 8-membered ring, which ring may beoptionally substituted with C₁₋₆ alkyl, aryl, C₁₋₆ alkoxy, or aryloxysubstituents;

R³ is H, C₁₋₆ alkyl, or C₁₋₆ alkoxy-substituted C₁₋₆ alkyl;

each of R⁴, R⁵, R⁶, and R⁷ individually is H, C₁₋₆ alkyl, or C₁₋₆alkoxy;

L¹ is a linker species selected from the group consisting of CR⁸R⁹,CR⁸R⁹CR¹⁰R¹¹, and O;

L² is a linker species selected from the group consisting of CH₂,CH₂CH₂, CH₂CH₂CH₂, or CH₂CH₂CH₂CH₂;

each of R⁸, R⁹, R¹⁰, and R¹¹ individually is hydrogen or C₁₋₆ alkyl; and

the dashed line indicates an optional double bond;

or a pharmaceutically acceptable salt thereof.

The present invention includes pharmaceutical compositions comprising acompound of the present invention or a pharmaceutically acceptable saltthereof. The pharmaceutical compositions of the present invention can beused for treating or preventing a wide variety of conditions ordisorders, and particularly those disorders characterized by dysfunctionof nicotinic cholinergic neurotransmission or the degeneration of thenicotinic cholinergic neurons.

The present invention Includes a method for treating or preventingdisorders and dysfunctions, such as CNS disorders and dysfunctions, andalso for treating or preventing certain conditions, for example,alleviating pain, hypertension, and inflammation, in mammals in need ofsuch treatment. The methods involve administering to a subject atherapeutically effective amount of a compound of the present invention,including a salt thereof, or a pharmaceutical composition that includessuch compounds.

DETAILED DESCRIPTION OF THE INVENTION I. Compounds

One embodiment of the present invention includes compounds of Formula I:

wherein

each of R¹ and R² individually is H, C₁₋₆ alkyl, or aryl-substitutedC₁₋₆ alkyl, or R¹ and R² combine with the nitrogen atom to which theyare attached to form a 3- to 8-membered ring, which ring may beoptionally substituted with C₁₋₆ alkyl, aryl, C₁, alkoxy, or aryloxysubstituents;

R³ is H, C₁₋₆ alkyl, or C₁₋₆ alkoxy-substituted C₁₋₆ alkyl;

each of R⁴, R⁵, R⁶, and R⁷ individually is H, C₁₋₈ alkyl, or C₁₋₆alkoxy;

L¹ is a linker species selected from the group consisting of CR⁸R⁹,CR⁸R⁹CR¹⁰R¹¹, and O;

L² is a linker species selected from the group consisting of CH₂,CH₂CH₂, CH₂CH₂CH₂, or CH₂CH₂CH₂CH₂;

each of R⁸, R⁹, R¹⁰, and R¹¹ individually is hydrogen or C₁₋₆ alkyl; and

the dashed line indicates an optional double bond:

or a pharmaceutically acceptable salt thereof.

In one embodiment, R¹ is H and R² is C₁₋₆ alkyl. In one embodiment, R³is C₁₋₆ alkyl. In one embodiment, each of R⁴, R⁵, R⁶, and R⁷ is H. Inone embodiment, L¹ is CR⁸R⁹, and each of R⁸ and R⁹ is hydrogen. In oneembodiment, L² is CH₂CH₂. In one embodiment, the dashed line is a singlebond.

One aspect of the present invention includes a pharmaceuticalcomposition comprising a compound of the present invention and apharmaceutically acceptable carrier.

One aspect of the present invention includes a method for the treatmentor prevention of a disease or condition mediated by a neuronal nicotinicreceptor, specifically through the use of non-competitive modulators(e.g., non-competitive antagonists), including but not limited channelblockers, comprising the administration of a compound of the presentinvention. In one embodiment, the disease or condition is a CNSdisorder. In another embodiment, the disease or condition isinflammation or an inflammatory response. In another embodiment, thedisease or condition Is pain. In another embodiment, the disease orcondition is neovascularization. In another embodiment, the disease orcondition is hypertension. In another embodiment, the disease orcondition is another disorder described herein.

One aspect of the present invention includes use of a compound of thepresent invention for the preparation of a medicament for the treatmentor prevention of a disease or condition mediated by a neuronal nicotinicreceptor, specifically through the use of non-competitive antagonists,such as channel blockers. In one embodiment, the disease or condition Isa CNS disorder. In another embodiment, the disease or condition isinflammation or an inflammatory response. In another embodiment, thedisease or condition is pain. In another embodiment, the disease orcondition is neovascularization. In another embodiment, the disease orcondition is hypertension. In another embodiment, the disease orcondition is another disorder described herein.

One aspect of the present invention includes a compound of the presentinvention for use as an active therapeutic substance. One aspect, thus,includes a compound of the present invention for use in the treatment orprevention of a disease or condition mediated by a neuronal nicotinicreceptor, specifically through the use of non-competitive antagonists,such as channel blockers. In one embodiment, the disease or condition isa CNS disorder. In another embodiment, the disease or condition isinflammation or an inflammatory response. In another embodiment, thedisease or condition is pain. In another embodiment, the disease orcondition is neovascularization. In another embodiment, the disease orcondition is hypertension. In another embodiment, the disease orcondition is another disorder described herein.

Particular diseases or conditions include depression, including majordepressive disorder, hypertension, irritable bowel syndrome (IBS),including IBS-D (diarrhea predominant), over active bladder (OAB), andaddiction, including smoking cessation.

The scope of the present invention includes all combinations of aspectsand embodiments.

The following definitions are meant to clarify, but not limit, the termsdefined. If a particular term used herein is not specifically defined,such term should not be considered Indefinite. Rather, terms are usedwithin their accepted meanings.

As used throughout this specification, the preferred number of atoms,such as carbon atoms, will be represented by, for example, the phrase“C, alkyl,” which refers to an alkyl group, as herein defined,containing the specified number of carbon atoms. Similar terminologywill apply for other preferred terms and ranges as well. Thus, forexample, C₁₋₆ alkyl represents a straight or branched chain hydrocarboncontaining one to six carbon atoms.

As used herein the term “alkyl” refers to a straight or branched chainhydrocarbon, which may be optionally substituted, with multiple degreesof substitution being allowed. Examples of “alkyl” as used hereininclude, but are not limited to, methyl, ethyl, propyl, isopropyl,isobutyl, n-butyl, tert-butyl, isopentyl, and n-pentyl.

As used herein, the term “alkylene” refers to a divalent group, such as“methylene,” “ethylene,” and “ethenylene,” which refer to divalent forms—CH₂—, —CH₂—CH₂—, and —CH═CH— respectively.

As used herein, the term “aryl” refers to a single benzene ring or fusedbenzene ring system which may be optionally substituted, with multipledegrees of substitution being allowed. Examples of “aryl” groups as usedInclude, but are not limited to, phenyl, 2-naphthyl, 1-naphthyl,anthracene, and phenanthrene. Preferable aryl rings have five- toten-members.

As used herein, a fused benzene ring system encompassed within the term“aryl” includes fused polycyclic hydrocarbons, namely where a cyclichydrocarbon with less than maximum number of noncumulative double bonds,for example where a saturated hydrocarbon ring (cycloalkyl, such as acyclopentyl ring) is fused with an aromatic ring (aryl, such as abenzene ring) to form, for example, groups such as indanyl andacenaphthylenyl, and also includes such groups as, for non-limitingexamples, dihydronaphthalene and tetrahydronaphthalene.

As used herein the term “alkoxy” refers to a group —OR^(a), where R^(a)is alkyl as herein defined.

As used herein the term “aryloxy” refers to a group —OR^(a), where R^(a)is aryl as herein defined.

As used herein “amino” refers to a group —NR^(a)R^(b), where each ofR^(a) and R^(b) is hydrogen. Additionally, “substituted amino” refers toa group —NR^(a)R^(b) wherein each of R^(a) and R^(b) individually isalkyl, arylalkyl or aryl. As used herein, when either R^(a) or R^(b) isother than hydrogen, such a group may be referred to as a “substitutedamino” or, for example if R^(a) is H and R^(b) is alkyl, as an“alkylamino.”

As used herein, the term “pharmaceutically acceptable” refers tocarrier(s), diluent(s), excipient(s) or salt forms of the compounds ofthe present invention that are compatible with the other ingredients ofthe formulation and not deleterious to the recipient of thepharmaceutical composition.

As used herein, the term “pharmaceutical composition” refers to acompound of the present invention optionally admixed with one or morepharmaceutically acceptable carriers, diluents, or excipients.Pharmaceutical compositions preferably exhibit a degree of stability toenvironmental conditions so as to make them suitable for manufacturingand commercialization purposes.

As used herein, the terms “effective amount”, “therapeutic amount”, and“effective dose” refer to an amount of the compound of the presentinvention sufficient to elicit the desired pharmacological ortherapeutic effects, thus resulting in an effective treatment of adisorder. Treatment of a disorder may be manifested by delaying orpreventing the onset or progression of the disorder, as well as theonset or progression of symptoms associated with the disorder. Treatmentof a disorder may also be manifested by a decrease or elimination ofsymptoms, reversal of the progression of the disorder, as well as anyother contribution to the well being of the patient.

The effective dose can vary, depending upon factors such as thecondition of the patient, the severity of the symptoms of the disorder,and the manner in which the pharmaceutical composition is administered.Typically, to be administered in an effective dose, compounds may beadministered in an amount of less than 5 mg/kg of patient weight. Thecompounds may be administered in an amount from less than about 1 mg/kgpatient weight to less than about 100 μg/kg of patient weight, andfurther between about 1 μg/kg to less than 100 μg/kg of patient weight.The foregoing effective doses typically represent that amount that maybe administered as a single dose, or as one or more doses that may beadministered over a 24 hours period.

The compounds of this invention may be made by a variety of methods,including well-established synthetic methods. Illustrative generalsynthetic methods are set out below and then specific compounds of theinvention are prepared in the working Examples.

In the examples described below, protecting groups for sensitive orreactive groups are employed where necessary in accordance with generalprinciples of synthetic chemistry. Protecting groups are manipulatedaccording to standard methods of organic synthesis (T. W. Green and P.G. M. Wuts (1999) Protecting Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, herein incorporated by reference with regardto protecting groups). These groups are removed at a convenient stage ofthe compound synthesis using methods that are readily apparent to thoseskilled in the art. The selection of processes as well as the reactionconditions and order of their execution shall be consistent with thepreparation of compounds of the present invention.

The present invention also provides a method for the synthesis ofcompounds useful as intermediates in the preparation of compounds of thepresent invention along with methods for their preparation.

The compounds can be prepared according to the methods described belowusing readily available starting materials and reagents. In thesereactions, variants may be employed which are themselves known to thoseof ordinary skill in this art but are not described in detail here.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. Compounds having the present structureexcept for the replacement of a hydrogen atom by a deuterium or tritium,or the replacement of a carbon atom by a ¹³C- or ¹⁴C-enriched carbon arewithin the scope of the invention. For example, deuterium has beenwidely used to examine the pharmacokinetics and metabolism ofbiologically active compounds. Although deuterium behaves similarly tohydrogen from a chemical perspective, there are significant differencesin bond energies and bond lengths between a deuterium-carbon bond and ahydrogen-carbon bond. Consequently, replacement of hydrogen by deuteriumin a biologically active compound may result in a compound thatgenerally retains its biochemical potency and selectivity but manifestssignificantly different absorption, distribution, metabolism, and/orexcretion (ADME) properties compared to its isotope-free counterpart.Thus, deuterium substitution may result in improved drug efficacy,safety, and/or tolerability for some biologically active compounds.

The compounds of the present invention may crystallize in more than oneform, a characteristic known as polymorphism, and such polymorphic forms(“polymorphs”) are within the scope of the present invention.Polymorphism generally can occur as a response to changes intemperature, pressure, or both. Polymorphism can also result fromvariations in the crystallization process. Polymorphs can bedistinguished by various physical characteristics known in the art suchas x-ray diffraction patterns, solubility, and melting point.

Certain of the compounds described herein contain one or more chiralcenters, or may otherwise be capable of existing as multiplestereoisomers. The scope of the present invention includes mixtures ofstereoisomers as well as purified enantiomers orenantiomerically/diastereomerically enriched mixtures. Also includedwithin the scope of the invention are the individual isomers of thecompounds represented by the formulae of the present invention, as wellas any wholly or partially equilibrated mixtures thereof. The presentinvention also includes the individual isomers of the compoundsrepresented by the formulas above as mixtures with Isomers thereof inwhich one or more chiral centers are inverted.

When a compound is desired as a single enantiomer, such may be obtainedby stereospecific synthesis, by resolution of the final product or anyconvenient intermediate, or by chiral chromatographic methods as areknown in the art. Resolution of the final product, an intermediate, or astarting material may be effected by any suitable method known in theart. See, for example, Stereochemistry of Organic Compounds(Wiley-Interscience, 1994).

The present invention Includes a salt or solvate of the compounds hereindescribed, including combinations thereof such as a solvate of a salt.The compounds of the present invention may exist in solvated, forexample hydrated, as well as unsolvated forms, and the present inventionencompasses all such forms.

Typically, but not absolutely, the salts of the present invention arepharmaceutically acceptable salts. Salts encompassed within the term“pharmaceutically acceptable salts” refer to non-toxic salts of thecompounds of this Invention.

Examples of suitable pharmaceutically acceptable salts include inorganicacid addition salts such as chloride, bromide, sulfate, phosphate, andnitrate; organic acid addition salts such as acetate, galactarate,propionate, succinate, lactate, glycolate, malate, tartrate, citrate,maleate, fumarate, methanesulfonate, p-toluenesulfonate, and ascorbate;salts with acidic amino acid such as aspartate and glutamate; alkalimetal salts such as sodium salt and potassium salt; alkaline earth metalsalts such as magnesium salt and calcium salt; ammonium salt; organicbasic salts such as trimethylamine salt, triethylamine salt, pyridinesalt, picoline salt, dicyclohexylamine salt, andN,N′-dibenzylethylenediamine salt; and salts with basic amino acid suchas lysine salt and arginine salt. The salts may be in some caseshydrates or ethanol solvates.

Those of skill in the art of organic chemistry will appreciate that morethan one systematic name can be given to many organic compounds. Thescope of the present invention should not be considered as lackingclarity due to the several potential naming conventions possible for thecompounds.

II. General Synthetic Methods

Those skilled in the art of organic synthesis will appreciate that thereexist multiple means of producing compounds of the present invention, aswell as means for producing compounds of the present invention which arelabeled with a radioisotope appropriate to various uses.

One means of producing compounds of the present invention is outlined inScheme 1 (see Synthetic Examples). Thus, norcamphor (2-norbornanone) canbe alkylated adjacent to the carbonyl functionality, using techniqueswell known to those of skill in the art of organic synthesis. Typically,treatment of the ketone with strong base (e.g., sodium hydride, sodiumalkoxide, sodium amide) to form an enolate Intermediate, followed bytreatment with an alkyl halide or sulfonate, is used for suchtransformations. Under certain conditions, the alkylation can beperformed with an α,ω-dihaloalkane (such as 1,3-dibromopropane), suchthat a spiro linkage is formed. While Scheme 1 shows the formation of aspirocyclobutane (Compound II), other ring sizes (e.g.,spirocyclopentane) are also accessible in this manner, by using otherα,ω-dihaloalkanes. The carbonyl functionality can subsequently beconverted Into an exocyclic methylene (Compound III), using Wittig (orequivalent) chemistry. Treatment of exo-methylene compounds withhydrogen cyanide (or similar reagents, such as thiocyanates), in thepresence of strong acid, can provide the corresponding tertiaryformamido compounds, in a process known as the Ritter reaction.Reduction of the formamido compound, using a hydride reducing agent,such as lithium aluminum hydride or sodium bis(methoxyethoxy)aluminumhydride, gives the corresponding secondary amine, Compound IV.

Alternatively, substituted 2-norbomanones can also be used as startingmaterials in the transformation outlined in Scheme 2. Thus, each ofD-camphor and L-camphor (both commercially available) can be transformedinto stereoisomers of Compound V. Other ketone starting materials canalso be used. For instance, the homolog of 2-norbornanone,bicyclo[2.2.2]octan-2-one, can be made by hydrogenation ofbicyclo[2,2,2]oct-5-en-2-one, which in turn can be made by proceduressimilar to those published by Kozikowski and Schmiesing, J. Org. Chem.48: 1000-1007 (1983), herein incorporated by reference with regard tosuch reaction. Similarly, 7-oxabicyclo[2.2.1]hept-5-en-2-one, producedas described by Black and Vogel, Helv. Chim. Acta 67: 1612 (1984),herein incorporated by reference with regard to such reaction, can behydrogenated to give 7-oxabicyclo[2.2.1]heptan-2-one. Each of theseketones is a potential starting material for transformations similar tothose shown in Schemes 1 and 2.

The spirocyclopropane functionality can be Installed using Simmons-Smithand similar chemistries. Thus, reaction of 3-methylene-2-norbornanonewith diiodomethane in the presence of zinc-copper couple givesspiro[bicyclo[2.2.1]heptane-2,1′-cyclopropan]-3-one, which can then betransformed into compounds of the resent invention utilizing reactionsalready described. Certain spirocyclopropane-containing compounds areknown in the literature and also serve as a starting point for synthesisof compounds of the present invention. See, for instance, Gream andPincombe, Aust. J. Chem. 27: 543-565 (1974), herein incorporated byreference with regard to such reaction.

Secondary amines, such as Compounds IV and V, can be converted intotertiary amines through the intermediacy of amides and carbamates. Thus,sequential treatment of such compounds with di-tert-butyl dicarbonateand lithium aluminum hydride will produce the corresponding N-methyltertiary amine.

The Incorporation of specific radioisotopes is also possible. Forexample, reductions of amides and carbamates with lithium aluminumdeuteride or lithium aluminum tritide reducing agents can produceN-trideuteromethyl or N-tritritiomethyl amines. Alternatively,generation of an amide or carbamate, in which the carbonyl carbon is an¹¹C, ¹³C, or ¹⁴C atom, followed by reduction with lithium aluminumhydride, will produce an amine with the ¹¹C, ¹³C, or ¹⁴C atom,respectively, incorporated. The incorporation of specific radioisotopesis often desirable in the preparation of compounds that are to be usedin a diagnostic setting (e.g., as imaging agents) or in functional andmetabolic studies.

III. Pharmaceutical Compositions

Although it is possible to administer the compound of the presentinvention in the form of a bulk active chemical, it is preferred toadminister the compound in the form of a pharmaceutical composition orformulation. Thus, one aspect the present invention includespharmaceutical compositions comprising one or more compounds of FormulaI and/or pharmaceutically acceptable salts thereof and one or morepharmaceutically acceptable carriers, diluents, or excipients. Anotheraspect of the invention provides a process for the preparation of apharmaceutical composition including admixing one or more compounds ofFormula I and/or pharmaceutically acceptable salts thereof with one ormore pharmaceutically acceptable carriers, diluents or excipients.

The manner in which the compound of the present invention isadministered can vary. The compound of the present invention ispreferably administered orally. Preferred pharmaceutical compositionsfor oral administration include tablets, capsules, caplets, syrups,solutions, and suspensions. The pharmaceutical compositions of thepresent invention may be provided in modified release dosage forms suchas time-release tablet and capsule formulations.

The pharmaceutical compositions can also be administered via injection,namely, intravenously, intramuscularly, subcutaneously,intraperitoneally, intraarterially, intrathecally, andintracerebroventricularly. Intravenous administration is a preferredmethod of injection. Suitable carriers for injection are well known tothose of skill in the art and include 5% dextrose solutions, saline, andphosphate buffered saline.

The formulations may also be administered using other means, forexample, rectal administration. Formulations useful for rectaladministration, such as suppositories, are well known to those of skillin the art. The compounds can also be administered by inhalation, forexample, in the form of an aerosol; topically, such as, in lotion form;transdermally, such as, using a transdermal patch (for example, by usingtechnology that is commercially available from Novartis and AlzaCorporation), by powder injection, or by buccal, sublingual, orIntranasal absorption.

Pharmaceutical compositions may be formulated in unit dose form, or inmultiple or subunit doses

The administration of the pharmaceutical compositions described hereincan be intermittent, or at a gradual, continuous, constant or controlledrate. The pharmaceutical compositions may be administered to awarm-blooded animal, for example, a mammal such as a mouse, rat, cat,rabbit, dog, pig, cow, or monkey; but advantageously is administered toa human being. In addition, the time of day and the number of times perday that the pharmaceutical composition is administered can vary.

The compounds of the present invention may be used in the treatment of avariety of disorders and conditions and, as such, may be used incombination with a variety of other suitable therapeutic agents usefulin the treatment or prophylaxis of those disorders or conditions. Thus,one embodiment of the present invention includes the administration ofthe compound of the present invention in combination with othertherapeutic compounds. For example, the compound of the presentinvention can be used in combination with other NNR ligands (such asvarenicline), allosteric modulators of NNRs, antioxidants (such as freeradical scavenging agents), antibacterial agents (such as penicillinantibiotics), antiviral agents (such as nucleoside analogs, likezidovudine and acyclovir), anticoagulants (such as warfarin),anti-inflammatory agents (such as NSAIDs), anti-pyretics, analgesics,anesthetics (such as used in surgery), acetylcholinesterase inhibitors(such as donepezil and galantamine), antipsychotics (such ashaloperidol, clozapine, olanzapine, and quetiapine), immuno-suppressants(such as cyclosporin and methotrexate), neuroprotective agents, steroids(such as steroid hormones), corticosteroids (such as dexamethasone,predisone, and hydrocortisone), vitamins, minerals, nutraceuticals,anti-depressants (such as imipramine, fluoxetine, paroxetine,escitalopram, sertraline, venlafaxine, and duloxetine), anxiolytics(such as alprazolam and buspirone), anticonvulsants (such as phenytoinand gabapentin), vasodilators (such as prazosin and sildenafil), moodstabilizers (such as valproate and aripiprazole), anti-cancer drugs(such as anti-proliferatives), antihypertensive agents (such asatenolol, clonidine, amlopidine, verapamil, and olmesartan), laxatives,stool softeners, diuretics (such as furosemide), anti-spasmotics (suchas dicyclomine), anti-dyskinetic agents, and anti-ulcer medications(such as esomeprazole). Such a combination of pharmaceutically activeagents may be administered together or separately and, when administeredseparately, administration may occur simultaneously or sequentially, inany order. The amounts of the compounds or agents and the relativetimings of administration will be selected in order to achieve thedesired therapeutic effect. The administration in combination of acompound of the present invention with other treatment agents may be incombination by administration concomitantly in: (1) a unitarypharmaceutical composition including both compounds; or (2) separatepharmaceutical compositions each including one of the compounds.Alternatively, the combination may be administered separately in asequential manner wherein one treatment agent is administered first andthe other second. Such sequential administration may be close in time orremote in time.

Another aspect of the present invention includes combination therapycomprising administering to the subject a therapeutically orprophylactically effective amount of the compound of the presentinvention and one or more other therapy including chemotherapy,radiation therapy, gene therapy, or immunotherapy.

IV. Method of Using Pharmaceutical Compositions

The compounds of the present invention can be used for the prevention ortreatment of various conditions or disorders for which other types ofnicotinic compounds have been proposed or are shown to be useful astherapeutics, such as CNS disorders, inflammation, inflammatory responseassociated with bacterial and/or viral infection, pain, metabolicsyndrome, autoimmune disorders, addictions, obesity or other disordersdescribed in further detail herein. This compound can also be used as adiagnostic agent (in vitro and in vivo). Such therapeutic and otherteachings are described, for example, in references previously listedherein, including Williams et al., Drug News Perspec. 7(4): 205 (1994),Arneric et al., CNS Drug Rev. 1(1): 1-26 (1995), Arneric et al., Exp.Opin. Invest. Drugs 5(1): 79-100 (1996), Bencherif et al., J. Pharmacol.Exp. Ther. 279: 1413 (1996), Lippiello et al., J. Pharmacol. Exp. Ther.279: 1422 (1996), Damaj et al., J. Pharmacol. Exp. Ther. 291: 390(1999); Chian et al., Anesthesiology 91: 1447 (1999), Lavand′homme andEisenbach, Anesthesiology 91: 1455 (1999), Holladay at al., J. Med.Chem. 40(28): 4169-94 (1997), Bannon et al., Science 279: 77 (1998), PCTWO 94/08992, PCT WO 96/31475, PCT WO 96/40682, and U.S. Pat. No.5,583,140 to Bencherif et al., U.S. Pat. No. 5,597,919 to Dull et al.,U.S. Pat. No. 5,604,231 to Smith et al, and U.S. Pat. No. 5,852,041 toCosford et al.

CNS Disorders

The compounds and their pharmaceutical compositions are useful in thetreatment or prevention of a variety of CNS disorders, includingneurodegenerative disorders, neuropsychiatric disorders, neurologicdisorders, and addictions. The compounds and their pharmaceuticalcompositions can be used to treat or prevent cognitive deficits anddysfunctions, age-related and otherwise; attentional disorders anddementias, including those due to infectious agents or metabolicdisturbances; to provide neuroprotection; to treat convulsions andmultiple cerebral infarcts; to treat mood disorders, compulsions andaddictive behaviors; to provide analgesia; to control inflammation, suchas mediated by cytokines and nuclear factor kappa B; to treatinflammatory disorders; to provide pain relief; and to treat infections,as anti-infectious agents for treating bacterial, fungal, and viralinfections. Among the disorders, diseases and conditions that thecompounds and pharmaceutical compositions of the present invention canbe used to treat or prevent are: age-associated memory impairment(AAMI), mild cognitive impairment (MCI), age-related cognitive decline(ARCD), pre-senile dementia, early onset Alzheimer's disease, seniledementia, dementia of the Alzheimer's type, Alzheimer's disease,cognitive impairment no dementia (CIND), Lewy body dementia,HIV-dementia, AIDS dementia complex, vascular dementia, Down syndrome,head trauma, traumatic brain injury (TBI), dementia pugilistica,Creutzfeld-Jacob Disease and prion diseases, stroke, central ischemia,peripheral ischemia, attention deficit disorder, attention deficithyperactivity disorder, dyslexia, schizophrenia, schizophreniformdisorder, schizoaffective disorder, cognitive dysfunction inschizophrenia, cognitive deficits in schizophrenia. Parkinsonismincluding Parkinson's disease, postencephalitic parkinsonism,parkinsonism-dementia of Gaum, frontotemporal dementia Parkinson's Type(FTDP), Pick's disease, Niemann-Pick's Disease, Huntington's Disease,Huntington's chorea, dyskinesia, tardive dyskinesia, spastic dystonia,hyperkinesia, progressive supranuclear palsy, progressive supranuclearparesis, restless leg syndrome, Creutzfeld-Jakob disease, multiplesclerosis, amyotrophic lateral sclerosis (ALS), motor neuron diseases(MND), multiple system atrophy (MSA), corticobasal degeneration,Guillain-Barré Syndrome (GBS), and chronic inflammatory demyelinatingpolyneuropathy (CIDP), epilepsy, autosomal dominant nocturnal frontallobe epilepsy, mania, anxiety, depression, including major depressivedisorder (MDD), premenstrual dysphoria, panic disorders, bulimia,anorexia, narcolepsy, excessive daytime sleepiness, bipolar disorders,generalized anxiety disorder, obsessive compulsive disorder, rageoutbursts, conduct disorder, oppositional defiant disorder, Tourette'ssyndrome, autism, drug and alcohol addiction, tobacco addiction and,thus, useful as an agent for smoking cessation, compulsive overeatingand sexual dysfunction.

Cognitive impairments or dysfunctions may be associated with psychiatricdisorders or conditions, such as schizophrenia and other psychoticdisorders, including but not limited to psychotic disorder,schizophreniform disorder, schizoaffective disorder, delusionaldisorder, brief psychotic disorder, shared psychotic disorder, andpsychotic disorders due to a general medical conditions, dementias andother cognitive disorders, including but not limited to mild cognitiveimpairment, pre-senile dementia, Alzheimer's disease, senile dementia,dementia of the Alzheimer's type, age-related memory impairment, Lewybody dementia, vascular dementia, AIDS dementia complex, dyslexia,Parkinsonism including Parkinson's disease, cognitive impairment anddementia of Parkinson's Disease, cognitive impairment of multiplesclerosis, cognitive impairment caused by traumatic brain injury,dementias due to other general medical conditions, anxiety disorders,Including but not limited to panic disorder without agoraphobia, panicdisorder with agoraphobia, agoraphobia without history of panicdisorder, specific phobia, social phobia, obsessive-compulsive disorder,post-traumatic stress disorder, acute stress disorder, generalizedanxiety disorder and generalized anxiety disorder due to a generalmedical condition, mood disorders, Including but not limited to majordepressive disorder, dysthymic disorder, bipolar depression, bipolarmania, bipolar I disorder, depression associated with manic, depressiveor mixed episodes, bipolar II disorder, cyclothymic disorder, and mooddisorders due to general medical conditions, sleep disorders, includingbut not limited to dyssomnia disorders, primary insomnia, primaryhypersomnia, narcolepsy, parasomnia disorders, nightmare disorder, sleepterror disorder and sleepwalking disorder, mental retardation, learningdisorders, motor skills disorders, communication disorders, pervasivedevelopmental disorders, attention-deficit and disruptive behaviordisorders, attention deficit disorder, attention deficit hyperactivitydisorder, feeding and eating disorders of infancy, childhood, or adults,tic disorders, elimination disorders, substance-related disorders,including but not limited to substance dependence, substance abuse,substance intoxication, substance withdrawal, alcohol-related disorders,amphetamine or amphetamine-like-related disorders, caffeine-relateddisorders, cannabis-related disorders, cocaine-related disorders,hallucinogen-related disorders, inhalant-related disorders,nicotine-related disorders, opolid-related disorders, phencyclidine orphencyclidine-like-related disorders, and sedative-, hypnotic- oranxiolytic-related disorders, personality disorders, including but notlimited to obsessive-compulsive personality disorder and impulse-controldisorders.

Cognitive performance may be assessed with a validated cognitive scale,such as, for example, the cognitive subscale of the Alzheimer's DiseaseAssessment Scale (ADAS-cog). One measure of the effectiveness of thecompounds of the present invention in improving cognition may Includemeasuring a patient's degree of change according to such a scale.

Regarding compulsions and addictive behaviors, the compounds of thepresent invention may be used as a therapy for nicotine addiction,including as an agent for smoking cessation, and for other brain-rewarddisorders, such as substance abuse including alcohol addiction, illicitand prescription drug addiction, eating disorders, including obesity,and behavioral addictions, such as gambling, or other similar behavioralmanifestations of addiction.

The above conditions and disorders are discussed in further detail, forexample, in the American Psychiatric Association: Diagnostic andStatistical Manual of Mental Disorders, Fourth Edition, Text Revision,Washington, D.C., American Psychiatric Association, 2000. This Manualmay also be referred to for greater detail on the symptoms anddiagnostic features associated with substance use, abuse, anddependence.

Inflammation

The nervous system, primarily through the vagus nerve, is known toregulate the magnitude of the innate immune response by inhibiting therelease of macrophage tumor necrosis factor (TNF). This physiologicalmechanism is known as the “cholinergic anti-inflammatory pathway” (see,for example, Tracey, “The Inflammatory Reflex,” Nature 420: 853-9(2002)). Excessive inflammation and tumor necrosis factor synthesiscause morbidity and even mortality in a variety of diseases. Thesediseases include, but are not limited to, endotoxemia, rheumatoidarthritis, osteoarthritis, psoriasis, asthma, atherosclerosis,idiopathic pulmonary fibrosis, and inflammatory bowel disease.Inflammatory conditions that can be treated or prevented byadministering the compounds described herein include, but are notlimited to, chronic and acute inflammation, psoriasis, endotoxemia,gout, acute pseudogout, acute gouty arthritis, arthritis, rheumatoidarthritis, osteoarthritis, allograft rejection, chronic transplantrejection, asthma, atherosclerosis, mononuclear-phagocyte dependent lunginjury, idiopathic pulmonary fibrosis, atopic dermatitis, chronicobstructive pulmonary disease, adult respiratory distress syndrome,acute chest syndrome in sickle cell disease, inflammatory bowel disease,irritable bowel syndrome, including diarrhea predominant IBS. Crohn'sdisease, ulcers, ulcerative colitis, acute cholangitis, aphthousstomatitis, cachexia, pouchitis, glomerulonephritis, lupus nephritis,thrombosis, and graft vs. host reaction.

Inflammatory Response Associated with Bacterial and/or Viral Infection

Many bacterial and/or viral infections are associated with side effectsbrought on by the formation of toxins, and the body's natural responseto the bacteria or virus and/or the toxins. As discussed above, thebody's response to infection often involves generating a significantamount of TNF and/or other cytokines. The over-expression of thesecytokines can result in significant injury, such as septic shock (whenthe bacteria is sepsis), endotoxic shock, urosepsis, viral pneumonitisand toxic shock syndrome.

Cytokine expression is mediated by NNRs, and can be inhibited byadministering agonists or partial agonists of these receptors. Thosecompounds described herein that are agonists or partial agonists ofthese receptors can therefore be used to minimize the inflammatoryresponse associated with bacterial infection, as well as viral andfungal infections. Examples of such bacterial infections includeanthrax, botulism, and sepsis. Some of these compounds may also haveantimicrobial properties. Furthermore, the compounds can be used in thetreatment of Raynaud's disease, namely viral-induced painful peripheralvasoconstriction.

These compounds can also be used as adjunct therapy in combination withexisting therapies to manage bacterial, viral and fungal infections,such as antibiotics, antivirals and antifungals. Antitoxins can also beused to bind to toxins produced by the infectious agents and allow thebound toxins to pass through the body without generating an inflammatoryresponse. Examples of antitoxins are disclosed, for example, in U.S.Pat. No. 6,310,043 to Bundle et al. Other agents effective againstbacterial and other toxins can be effective and their therapeutic effectcan be complemented by co-administration with the compounds describedherein.

Pain

The compounds can be administered to treat and/or prevent pain,including acute, neurologic, Inflammatory, neuropathic and chronic pain.The compounds can be used in conjunction with opiates to minimize thelikelihood of opiate addiction (e.g., morphine sparing therapy). Theanalgesic activity of compounds described herein can be demonstrated inmodels of persistent inflammatory pain and of neuropathic pain,performed as described in U.S. Published Patent Application No.20010056084 A1 (Algeler at al.) (e.g., mechanical hyperalgesia in thecomplete Freund's adjuvant rat model of inflammatory pain and mechanicalhyperalgesia in the mouse partial sciatic nerve ligation model ofneuropathic pain).

The analgesic effect is suitable for treating pain of various genesis oretiology, in particular in treating inflammatory pain and associatedhyperalgesia, neuropathic pain and associated hyperalgesia, chronic pain(e.g., severe chronic pain, post-operative pain and pain associated withvarious conditions including cancer, angina, renal or biliary colic,menstruation, migraine, and gout). Inflammatory pain may be of diversegenesis, including arthritis and rheumatoid disease, teno-synovitis andvasculitis. Neuropathic pain includes trigeminal or herpetic neuralgia,neuropathies such as diabetic neuropathy pain, causalgia, low back painand deafferentation syndromes such as brachial plexus avulsion.

Neovascularization

Inhibition of neovascularization, for example, by administeringantagonists (or at certain dosages, partial agonists) of nicotinicreceptors can treat or prevent conditions characterized by undesirableneovascularization or angiogenesis. Such conditions can include thosecharacterized by inflammatory angiogenesis and/or ischemia-inducedangiogenesis. Neovascularization associated with tumor growth can alsobe Inhibited by administering those compounds described herein thatfunction as antagonists or partial agonists of nicotinic receptors.

Specific antagonism of nicotinic receptors reduces the angiogenicresponse to inflammation, ischemia, and neoplasia. Guidance regardingappropriate animal model systems for evaluating the compounds describedherein can be found, for example, in Heeschen. C. et al., “A novelangiogenic pathway mediated by non-neuronal nicotinic acetylcholinereceptors,” J. Clin. Invest. 110(4):527-36 (2002).

Representative tumor types that can be treated using the compoundsdescribed herein include SCLC, NSCLC, ovarian cancer, pancreatic cancer,breast carcinoma, colon carcinoma, rectum carcinoma, lung carcinoma,oropharynx carcinoma, hypopharynx carcinoma, esophagus carcinoma,stomach carcinoma, pancreas carcinoma, liver carcinoma, gallbladdercarcinoma, bile duct carcinoma, small intestine carcinoma, urinary tractcarcinoma, kidney carcinoma, bladder carcinoma, urothelium carcinoma,female genital tract carcinoma, cervix carcinoma, uterus carcinoma,ovarian carcinoma, choriocarcinoma, gestational trophoblastic disease,male genital tract carcinoma, prostate carcinoma, seminal vesiclescarcinoma, testes carcinoma, germ cell tumors, endocrine glandcarcinoma, thyroid carcinoma, adrenal carcinoma, pituitary glandcarcinoma, skin carcinoma, hemangiomas, melanomas, sarcomas, bone andsoft tissue sarcoma, Kaposi's sarcoma, tumors of the brain, tumors ofthe nerves, tumors of the eyes, tumors of the meninges, astrocytomas,gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas,Schwannomas, meningiomas, solid tumors arising from hematopoieticmalignancies (such as leukemias, chloromas, plasmacytomas and theplaques and tumors of mycosis fungoides and cutaneous T-celllymphoma/leukemia), and solid tumors arising from lymphomas.

The compounds can also be administered in conjunction with other formsof anti-cancer treatment, including co-administration withantineoplastic antitumor agents such as cis-platin, adriamycin,daunomycin, and the like, and/or anti-VEGF (vascular endothelial growthfactor) agents, as such are known in the art.

The compounds can be administered in such a manner that they aretargeted to the tumor site. For example, the compounds can beadministered in microspheres, microparticles or liposomes conjugated tovarious antibodies that direct the microparticles to the tumor.Additionally, the compounds can be present in microspheres,microparticles or liposomes that are appropriately sized to pass throughthe arteries and veins, but lodge in capillary beds surrounding tumorsand administer the compounds locally to the tumor. Such drug deliverydevices are known in the art.

Other Disorders

In addition to treating CNS disorders, inflammation, andneovascularization, and pain, the compounds of the present invention canbe also used to prevent or treat certain other conditions, diseases, anddisorders in which NNRs play a role. Examples Include autoimmunedisorders such as lupus, disorders associated with cytokine release,cachexia secondary to infection (e.g., as occurs in AIDS, AIDS relatedcomplex and neoplasia), obesity, pemphitis, urinary incontinence,overactive bladder (OAB), diarrhea, constipation, retinal diseases,infectious diseases, myasthenia, Eaton-Lambert syndrome, hypertension,preeclampsia, osteoporosis, vasoconstriction, vasodilatation, cardiacarrhythmias, type I diabetes, type II diabetes, bulimia, anorexia andsexual dysfunction, as well as those indications set forth in publishedPCT application WO 98/25619. The compounds of this invention can also beadministered to treat convulsions such as those that are symptomatic ofepilepsy, and to treat conditions such as syphilis and Creutzfeld-Jakobdisease.

Compounds of the present invention may be used to treat bacterialinfections and dermatologic conditions, such as pemphigus folliaceus,pemphigus vulgaris, and other disorders, such as acantholysis, whereautoimmune responses with high ganglionic NNR antibody titer is present.In these disorders, and in other autoimmune diseases, such as MystheniaGravis, the fab fragment of the antibody binds to the NNR receptor(crosslinking 2 receptors), which induces internalization anddegradation.

Diagnostic Uses

The compounds can be used in diagnostic compositions, such as probes,particularly when they are modified to include appropriate labels. Forthis purpose the compounds of the present invention most preferably arelabeled with the radioactive Isotopic moiety ¹¹C.

The administered compounds can be detected using position emissiontopography (PET). A high specific activity is desired to visualize theselected receptor subtypes at non-saturating concentrations. Theadministered doses typically are below the toxic range and provide highcontrast images. The compounds are expected to be capable ofadministration in non-toxic levels. Determination of dose is carried outin a manner known to one skilled in the art of radiolabel imaging. See,for example, U.S. Pat. No. 5,969,144 to London at al.

The compounds can be administered using known techniques. See, forexample, U.S. Pat. No. 5,969,144 to London et al, as noted. Thecompounds can be administered in formulation compositions thatincorporate other ingredients, such as those types of ingredients thatare useful in formulating a diagnostic composition. Compounds useful inaccordance with carrying out the present invention most preferably areemployed in forms of high purity. See, U.S. Pat. No. 5,853,696 toElmalch et al.

After the compounds are administered to a subject (e.g., a humansubject), the presence of that compound within the subject can be imagedand quantified by appropriate techniques in order to indicate thepresence, quantity, and functionality. In addition to humans, thecompounds can also be administered to animals, such as mice, rats, dogs,and monkeys. SPECT and PET Imaging can be carried out using anyappropriate technique and apparatus. See Villemagne et al., In: Arnericet al. (Eds.) Neuronal Nicotinic Receptors: Pharmacology and TherapeuticOpportunities, 235-250 (1998) and U.S. Pat. No. 5,853,696 to Elmalch etal., each herein incorporated by reference, for a disclosure ofrepresentative imaging techniques.

V. Synthetic Examples Example 1:Exo-N,3-dimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-amine

To a solution of 2-norbornanone (norcamphor) (16.0 g, 145 mmol) and1,3-dibromopropane (203 mmol, 20.7 mL; 41.1 g) in diethyl ether (450 mL)was added sodium amide (363 mmol, 14.8 g) and the mixture was stirred atreflux for 24 h. The mixture was poured into 200 mL of ice-water, andthe organic layer was separated. The aqueous layer was extracted with200 mL of ether. The combined ether extracts were concentrated, and theliquid residue was distilled at 60-100° C. at 10-20 Torr vacuum toobtain 14 g of impure product. This was dissolved in 150 mL of hexanesand stirred with a solution of potassium permanganate (12.0 g, 75.9mmol) in water (150 mL) for 5 h. The biphasic mixture was filteredthrough a bed of diatomaceous earth, which was then washed with hexanes(100 mL). The hexane layer was separated, and the aqueous layer wasextracted with 600 mL of hexanes. The hexane layers were combined,concentrated, and purified on silica gel column, eluting with 10-40%ether in hexanes, to obtainspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-one (Compound II inScheme 1) (6.1 g, 28% yield) as oil. ¹H NMR (CDCl₃, 400 MHz): δ2.55-2.49 (m, 2H), 2.18-2.08 (m, 2H), 2.00-1.58 (m, 7H), 1.49-1.36 (m,3H); LCMS (m/z): 151 (M+1).

To a suspension of (methyl)triphenylphosphonium bromide (49.9 mmol, 18.2g) in dry tetrahydrofuran (THF) (100 mL) at −78° C. was addedn-butyllithium (46.5 mmol, 18.6 mL of 2.5 M solution in hexanes). Themixture was stirred for 30 min at −78° C. To this mixture was addedspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-one (5.00 g, 33.3 mmol).The resulting mixture was stirred at ambient temperature for 20 h.Hexanes (300 mL) were added, and the mixture was filtered. The filtratewas concentrated, and the residue was purified on an 80 g silica gelcolumn, eluting with hexanes, to obtain3-methylenespiro[bicyclo[2.2.1]heptane-2,1′-cyclobutane](Compound III inScheme 1) (3.7 g, 75%) as oil. ¹H NMR (CDCl₃, 400 MHz): δ 4.82 (s, 2H),2.63 (brs, 1H), 2.22 (bra, 1H), 2.05-1.78 (m, 6H), 1.63-1.52 (m, 1H),1.48-1.34 (m, 3H), 1.21-1.12 (m, 2H).

To a suspension of3-methylenespiro[bicyclo[2.2.1]heptane-2,1′-cyclobutane] (2.10 g, 14.2mmol) and potassium thiocyanate (14.2 mmol, 1.39 g) was slowly added asolution of sulfuric acid (1.40 g; 14.3 mmol) in water (0.52 mL) over 15min at 50° C. The solution was stirred at 85° C. for 5.5 h. The solutionwas cooled to ambient temperature, diluted with toluene (20 mL), andwashed sequentially with water (20 mL) and saturated aqueous sodiumbicarbonate (10 mL). The toluene layer was collected, dried overanhydrous sodium sulfate, and filtered. To the filtrate was added sodiumbis(methoxyethoxy)aluminum hydride (28 mmol, 7.9 mL of 65-70% solutionin toluene), and the resulting mixture was stirred at 85° C. for 2 h.The mixture was cooled to 0° C. and a mixture of 3N aqueous sodiumhydroxide (3 mL) and 5% sodium hypochlorite (15 mL) was slowly addeddrop-wise, in intervals. The toluene layer was separated and washed withwater (30 mL). The toluene layer was then extracted with 1 N aqueoushydrochloric acid (2×10 mL). The toluene layer was discarded, and thecombined hydrochloric acid extracts were made basic by addition of 10%aqueous sodium hydroxide (to pH10). The basic aqueous mixture wasextracted with ether (2×30 mL). The ether extracts were collected,concentrated, and purified by silica gel column chromatography, elutingwith 0-40% CMA (chloroform:methenol:30% aqueous ammonia, 9:1:0.1) inchloroform, to obtainexo-N,3-dimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-amine(0.38 g, 15% yield) (Compound IV in Scheme 1) as oil. The oil wasdissolved in 5 mL of dichloromethane, cooled in ice-bath, and combinedwith 2 mL of 6 M aqueous hydrochloric acid. The mixture was concentratedand vacuum dried to obtain the hydrochloride salt. ¹H NMR (D₂O, 400MHz): δ 2.41 (s, 3H), 2.24-2.18 (m, 2H), 1.98-1.90 (m, 1H), 1.82-1.74(m, 1H), 1.67-1.58 (m, 2H), 1.52-1.11 (m, 8H), 0.95 (s, 3H); LCMS (m/z):180 (M+1).

The exo stereochemistry was established by NMR.

Example 2: Chiral chromatographic separation ofexo-N,3-dimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-amine

Exo-N,3-dimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-amine(2.0 g) was dissolved in 20 mL of acetonitrile and was separated with0.2 mL injections on chiral column (Chiral Pak AD-H, 5 micron, 250×20cm), using 0.2% diethylamine in acetonitrile/isopropanol (95:5), with aflow rate of 10 mL/min. Fractions containing peak 1 (early eluting) andpeak 2 (late eluting) were separately concentrated. The two residueswere individually dissolved in 10 mL of dichloromethane, treated with 2mL of 6N aqueous hydrochloric acid, and concentrated to dryness. Thesehydrochloride salt products weighed 0.74 g (peak 1) and 0.48 g (peak 2),respectively.

Example 3:N,3-dimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclopentan]-3-amine

To a solution of 2-norbornanone (25.0 g, 227 mmol) and 1,4-dibromobutane(68.0 g, 317 mmol) in diethyl ether (700 mL) was added sodium amide(23.1 g, 567 mmol). This mixture was heated at reflux for 24 h, cooledand poured into 200 mL of ice-water. The organic layer was collected,and the aqueous layer was extracted with 200 mL of diethyl ether. Thecombined diethyl ether extracts were concentrated, and the residue wasdistilled at 65-80° C. at 7-15 Torr to obtain 19 g of impure product.This was dissolved in hexanes (500 mL) and stirred with aqueouspotassium permanganate (30 g, 0.19 mol, in 500 mL) for 5 h. The mixturewas filtered, and the hexane layer was collected. The aqueous layer wasextracted with 600 mL of hexanes. The combined hexane layers wereconcentrated, and the residue purified on a silica gel column, elutingwith 5-15% ethyl acetate in hexanes, to obtainspiro[bicyclo[2.2.1]heptane-2,1′-cyclopentan]-3-one (12.6 g, 33.8%) asoil. ¹H NMR (CDCl₃, 400 MHz): δ 2.56 (d, J=5.1 Hz, 1H), 2.24 (bs, 1H),1.44-1.886 (m, 14H); LCMS (m/z): 165 (M+1).

To a suspension of (methyl)triphenylphosphonium bromide (17.6 g, 48.4mmol) in THF (100 mL) at −78° C. was added n-butyllithium (18.1 mL of2.5 M solution in THF, 45 mmol) and the mixture was stirred 30 min. Tothis mixture was addedspiro[bicyclo[2.2.1]heptane-2,1′-cyclopentan]-3-one (5.30 g, 32.3 mmol),and the reaction was stirred at ambient temperature for 18 h. Hexanes(200 mL) were added, and the mixture was filtered. The filtrate wasconcentrated, and the residue was purified on an 80 g silica gel column,eluting with hexanes, to obtain3-methylenespiro[bicyclo[2.2.1]heptane-2,1′-cyclopentane] (4.80 g,91.7%) as oil.

Sulfuric acid (1.81 mL, 2.96 g, 30.2 mmol) was slowly added to asuspension of 3-methylenespiro[bicyclo[2.2.1]heptane-2,1′-cyclopentane](4.80 g, 29.6 mmol) and potassium thiocyanate (2.96 g, 30.2 mmol) at 50°C. The reaction mixture was then stirred at 85° C. for 5.5 h. cooled toambient temperature, diluted with toluene (30 mL) and washed with water(20 mL) followed by with saturated aqueous sodium bicarbonate (10 mL).The toluene layer was dried over anhydrous sodium sulfate and filtered.To the filtrate was added sodium bis(methoxyethoxy)aluminum hydride (40%solution in toluene, 2 equivalents) and the reaction was stirred at 85°C. for 2 h. The reaction was cooled to 0° C., and a solution of 3Naqueous sodium hydroxide (20 mL) in 5% aqueous sodium hypochlorite (35mL) was slowly added (drop-wise at intervals). The toluene layer wasseparated and washed with water (30 mL). The toluene layer was thenextracted with 1N aqueous hydrochloric acid (2×10 mL) and discarded. Theaqueous hydrochloric acid layer was made basic (to pH 10) by addition of10% aqueous sodium hydroxide and extracted with diethyl ether. Thediethyl ether extracts were concentrated, and the residue was purifiedby silica gel column chromatography, using 0-40% CMA(chloroform:methanol:30% aqueous ammonia, 9:1:0.1) in chloroform toobtain N,3-dimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclopentan]-3-amine(1.2 g, 53%) as oil. ¹H NMR (CDCl₃, 400 MHz): δ 2.28 (s, 3H), 2.24 (bs,1H), 1.84-1.76 (m, 2H), 1.73-1.68 (m, 1H), 1.62-1.52 (m, 4H), 1.48-1.24(m, 7H), 1.09-1.05 (m, 1H), 1.04 (s, 3H); LCMS (m/z): 194 (M+1).

Example 4: General procedure for making N-substitutedspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-amines

Certain N-substitutedspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan-3-amines can be prepared bythe reductive amination ofspiro[bicyclo(2.2.1]heptane-2,1′-cyclobutan]-3-one. The followingprocedure, utilizing methylamine and providingN-methylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan)-3-aminetrifluoroacetate, is exemplary. Reductive aminations utilizingdimethylamine, azetidine, and pyrrolidine were performed in a similarfashion.

To a solution of spiro(bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-one(0.15 g, 1.0 mmol) and methylamine (4.0 mL of 2.0 M solution in THF, 8.0mmol) in 1,2-dichloroethane (10 mL) was added acetic acid (0.2 mL) andsodium triacetoxyborohydride (0.85 g, 4.0 mmol). The reaction wasstirred at ambient temperature for 48 h, diluted with dichloromethane(10 mL), washed with saturated aqueous sodium bicarbonate solution (10mL), and concentrated. The residue was purified on preparative HPLC,eluting with mixtures of 0.05% formic acid in water and 0.05% formicacid in acetonitrile. Selected fractions were concentrated, and theresidue was dissolved in methanol (2 mL). Trifluoroacetic acid (0.1 mL)was added, and the mixture was concentrated and vacuum dried to obtainN-methylspiro[bicyclo[2.2.]heptane-2,1′-cyclobutan]-3-aminetrifluoroacetate (0.088 g) as gum. ¹H NMR (CD₃OD, 400 MHz): δ 3.06-3:02(m, 1H), 2.568 (s, 3H), 2.54 (brs, 1H), 2.34 (brs, 1H), 2.02-1.83 (m,6H), 1.56-1.42 (m, 5H), 1.26-1.32 (m, 1H); LCMS (m/z): 166 (M+1).

Example 5:(1S,3R,4R)—N,4,7,7-Tetramethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-aminehydrochloride and(1S,3S,4R)—N,4,7,7-tetramethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-aminehydrochloride

The following chemistry, using D-camphor as a starting material, wasrepeated using L-camphor as a starting material, yielding products thatare enantiomeric to those described here.

A mixture of D-(+)-camphor (4.40 g, 28.9 mmol) and sodium amide (2.50 g,61.5 mmol) in toluene (100 mL) was stirred at 100° C. for 30 min. Asolution of 1,3-dibromopropane (31.8 mmol, 3.24 mL, 6.42 g) in toluene(20 mL) was added, and the reaction was heated at reflux for 3 h. Thereaction was cooled to ambient temperature, washed with water (100 mL),dried over anhydrous sodium sulfate and concentrated. The residue wasdissolved in 5% methanol in dichloromethane (80 mL) and cooled to −78°C. Ozone was passed through the solution until the blue color persisted(˜10 minutes). Dimethyl sulfide (2 mL) was then added, and the reactionwas warmed slowly to ambient temperature. The reaction mixture wasconcentrated, and the residue was purified on a silica gel column (40g), eluting with 0-20% ether in hexanes, to obtain(1S,4R)-4,7,7-trimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-one(1.66 g, 29.9% yield) as oil. ¹H NMR (CDCl₃, 400 MHz): δ 2.26 (m, 1H),2.10-1.97 (m, 5H), 1.85-1.1.66 (m, 2H), 1.62-1.53 (m, 1H), 1.47-1.40 (m,1H), 1.28-1.19 (m, 1H), 0.94 (s, 3H), 0.88 (s, 3H), 0.75 (s, 3H); LCMS(m/z): 193 (M+1).

A mixture of(1S,4R)-4,7,7-trimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-one(1.60 g, 8.32 mmol) and formamide (10 mL) in formic acid (7 mL) wasstirred at 175° C. for 72 h. The reaction mixture was cooled to ambienttemperature, poured into 200 mL of ice-water, and extracted with ether(2×50 mL). The combined ether extracts were washed with water (40 mL),dried over anhydrous sodium sulfate and concentrated to obtainN-(4,7,7-trimethylspiro[bicycle[2.2.1]heptane-2,1′-cyclobutan]-3-yl)formamide(1.55 g, 84.2% yield) as gum.

To a solution ofN-(4,7,7-trimethylspiro[bicyclo(2.2.1]heptane-2,1′-cyclobutan]-3-yl)formamide(1.50 g, 6.78 mmol) in THF (40 mL) at 0° C. was slowly added lithiumaluminum hydride (27.1 mmol, 27.1 mL of 1.0 M solution in THF). Aftercomplete addition, the reaction was refluxed for 48 h. The reactionmixture was cooled to 0° C. and quenched by addition, in portions, ofsold sodium sulfate decahydrate (10 g). After stirring for 1 h, thismixture was filtered, and the filtrate was concentrated. The residue waspurified on a 40 g silica gel column, using 0-100% CMA(chloroform:methanol:30% aqueous ammonia; 9:1:0.1) in chloroform as theeluent, to obtain an exo-amine product,(1S,3R,4R)—N,4,7,7-tetramethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-amine(0.49 g; 35% yield), and an endo-amine product,(1S,3S,4R)—N,4,7,7-tetramethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-amine(0.30 g; 21% yield), both as oils. The two products were converted totheir hydrochloride salts by dissolving each in 1 mL of concentratedhydrochloric acid and concentrating and vacuum drying the samples. ¹HNMR ofexo-(1S,3R,4R)—N,4,7,7-tetramethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-aminehydrochloride (D₂O, 400 MHz): δ 2.80 (s, 3H), 2.72 (brs, 1H), 2.22-1.91(m, 4H), 1.84-1.72 (m, 3H), 1.54-1.45 (m, 2H), 1.38-1.31 (m, 1H),1.10-1.01 (m, 1H), 0.87 (s, 3H), 0.75 (s, 3H), 0.72 (s, 3H); LCMS (m/z):208 (M+1). ¹H NMR ofendo-(1S,3S,4R)—N,4,7,7-tetramethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-aminehydrochloride (D₂O, 400 MHz): δ 3.12 (brs, 1H), 2.79 (s, 3H), 2.26-2.16(m, 1H), 2.01-1.85 (m, 3H), 1.78-1.69 (m, 3H), 1.60-1.51 (m, 1H),1.36-1.24 (m, 2H), 1.08-1.00 (m, 1H), 0.85 (s, 3H), 0.78 (s, 3H), 0.75(s, 3H); LCMS (m/z): 208 (M+1).

Example 6: General Procedure for Converting Secondary Amines intoN-Methyl Tertiary Amines

Certain N-methyl tertiary amines can be prepared by the reductiveamination of the corresponding secondary amines. The followingprocedure, utilizing formaldehyde and providingexo-(1S,3R,4R)—N,N,4,7,7-pentamethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-aminehydrochloride, is exemplary. Analogous N-methylation reactions werecarried out on a variety of secondary amines.

To a solution of(1S,3R,4R)—N,4,7,7-tetramethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-amine(0.10 g, 0.48 mmol) and 30% aqueous formaldehyde (1 mL) in methanol (4mL) was added sodium triacetoxyborohydride (0.31 g, 1.4 mmol), and thereaction was stirred at ambient temperature for 16 h. The reaction wasquenched with saturated aqueous sodium bicarbonate solution (30 mL) andextracted with dichloromethane (2×30 mL). Formic acid (0.2 mL) was addedto the combined organic extracts and they were concentrated on rotaryevaporator. The residue was purified by preparative LCMS, using mixturesof 0.05% formic acid in water and 0.05% formic acid in acetonitrile.Selected fractions were combined, made basic (pH 9) by addition of 10%aqueous sodium hydroxide and extracted with dichloromethane (2×30 mL).The combined organic extracts were treated with 0.5 mL of concentratedhydrochloric acid. This mixture was concentrated and vacuum dried toobtain(1S,3R,4R)—N,N,4,7,7-pentamethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-aminehydrochloride (0.06 g) as white solid. ¹H NMR (D₂O, 400 MHz): δ 3.27 (s,3H), 3.19 (s, 3H), 3.10 (s, 1H), 2.55-2.28 (m, 4H), 2.18-1.97 (m, 3H),1.80-1.60 (m, 3H), 1.45-1.36 (m, 1H), 1.28 (s, 3H), 1.02 (s, 3H), 1.01(s, 3H); LCMS (m/z): 222 (M+1).

Example 7: N-Methylspiro[bicyclo[2.2.1]heptane-2,1′-cyclopropan]-3-aminetrifluoroacetate

Neat 3-methylene-2-norbornanone (8.9 g, 73 mmol), followed by neatdiiodomethane (8.30 mL, 103 mmol), were added to a slurry of zinc-coppercouple (9.1 g, 57 mmol) In diethyl ether (75 mL). The resulting mixturewas heated at reflux for 6 h. A second portion of zinc-copper couple (10g) was added, and reflux was continued for an additional 16 h. Thereaction was then quenched with water (200 mL) and diluted with diethylether (200 mL). The biphasic mixture was filtered through a pad ofdiatomaceous earth. The organic layer was separated, washed with 10%aqueous hydrochloric acid (2×50 mL), dried over anhydrous magnesiumsulfate and concentrated. The residue was passed through a silica gelcolumn, eluting with dichloromethane. Selected fractions wereconcentrated, and the residue was vacuum distilled on a bulb-to-bulbdistillation apparatus at 3 Torr, collectingspiro[bicyclo[2.2.1]heptane-2,1′-cyclopropan-3-one (1.6 g) as oil. ¹HNMR (CDCl₃, 400 MHz): δ 2.71-2.69 (m, 1H), 2.06 (bra, 1H), 2.00-1.72 (m,3H), 1.66-1.57 (m, 3H), 1.09-1.00 (m, 2H), 0.91-0.89 (m, 1H), 0.80-0.75(m, 1H).

To a solution of spiro[bicyclo(2.2.1]heptane-2,1′-cyclopropan]-3-one(0.13 g, 0.96 mmol) and methylamine (4.0 mL of 2.0 M solution in THF,8.0 mmol) in 1,2-dichloroethane (10 mL) was added acetic acid (0.2 mL)and sodium triacetoxyborohydride (0.85 g, 4.0 mmol) and the reaction wasstirred at ambient temperature for 48 h. The reaction was diluted withdichloromethane (10 mL), washed with saturated aqueous sodiumbicarbonate solution (10 mL), and concentrated. The residue was purifiedon preparative HPLC, eluting with mixtures of 0.05% formic acid in waterand 0.05% formic acid in acetonitrile. Selected fractions wereconcentrated, and the residue was dissolved in methanol (2 mL).Trifluoroacetic acid (0.1 mL) was added, and the mixture wasconcentrated and vacuum dried, to obtainN-methylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-aminetrifluoroacetate (0.005 g) as gum. ¹H NMR (CD₃OD, 400 MHz): δ 2.76 (brs,1H), 2.59 (s, 3H), 1.85-1.82 (m, 1H), 1.69-1.55 (m, 6H), 1.34-1.28 (m,1H), 0.83-0.78 (m, 1H), 0.67-0.62 (m, 1H), 0.58-0.50 (m, 2H); LCMS(m/z): 152 (M+1).

Example 8:N,3-dimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclopropan]-3-aminehydrochloride

To a solution of(3-methylspiro[bicyclo[2.2.1]hept[5]ene-2,1′-cyclopropane]-3-yl)methanol(9.4 g, 57 mmol), prepared as described by Gream and Pincombe, Aust. J.Chem. 27: 543-565 (1974), herein incorporated by reference with regardto such preparation, in methanol (20 mL) was added 0.8 g 10% Pd/C (wet)under nitrogen. The atmosphere was replaced with hydrogen (50 psi), andthe mixture was shaken for 4 h at ambient temperature. The reaction wasthen filtered through a pad of diatomaceous earth, which was then washedwith methanol. The filtrate was concentrated to yield 9.60 g of(3-methylspiro[bicyclo[2.2.1]heptane-2,1′-cyclopropane]-3-yl)methanol asa white solid (99%).

To a stirred solution of chromium trioxide (8.0 g, 76 mmol) in water (30mL) cooled in an ice bath, was added carefully 96% sulfuric acid (6.9mL, 120 mmol). While continuing to cool and stir the oxidant solution inan ice bath, a solution of (3-methylspiro[bicyclo[2.2.1]heptane-2,1′-cyclopropane]-3-yl)methanol (9.5 g; 57 mmol)In acetone (115 mL) was added over a 20 min period. After completeaddition, the reaction mixture was stirred for 3 h while warming toambient temperature. The reaction was then diluted with water (45 mL)and ethyl acetate (200 mL). Slowly, sodium bisulfite powder was addeduntil the brown color dissipated and the aqueous layer became blue. Thephases were then separated, and the aqueous layer was washed with ethylacetate (2×100 mL). The organic layers were combined and dried overanhydrous magnesium sulfate. The drying agent was removed by filtration,and the filtrate was concentrated to yield a green oil. The oil waspurified by silica gel (200 g) column chromatography, using a 0-50%ethyl acetate in hexanes gradient. Selected fractions were combined andconcentrated to yield 6.0 g of3-methylspiro[bicyclo[2.2.1]heptane-2,1′-cyclopropane]-3-carboxylic acidas a white solid (58%).

To a stirred solution of3-methylspiro[bicyclo[2.2.1]heptane-2,1′-cyclopropane]-3-carboxylic acid(2.8 g, 15 mmol) and triethylamine (2.6 mL, 18 mmol) in toluene (70 mL)cooled in an ice bath, was added diphenylphosphonic azide (3.5 mL, 16mmol). The reaction mixture was warmed to 90° C. and stirred for 2.5 h.Benzyl alcohol (1.7 mL, 16 mmol) was then added to the reaction, and themixture was stirred an additional 16 h at 90° C. The reaction mixturewas cooled and concentrated. The residue was purified by silica gel (60g) column chromatography, using a 0-15% ethyl acetate in hexanesgradient. Selected fractions were combined and concentrated to yield 1.6g of a mixture of materials, including 3-isocyanato-3-methylspiro[bicyclo[2.2.1]heptane-2,1′-cyclopropane] and the corresponding benzylcarbamate, as a white solid. This mixture was dissolved in dry THF (16mL) and cooled in an ice bath. Lithium aluminum hydride (8.5 mL of 2.0Min THF, 17 mmol) was slowly added. The reaction was warmed to 55° C. for3 h. The reaction was then cooled in an ice-bath and diluted withdiethyl ether (20 ml.). The reaction was quenched by careful addition ofwater until gas evolution subsided. The resulting viscous white slurrywas stirred at ambient temperature for 1 h, during which time the saltsbecame more granular. The slurry was then filtered through a pad ofdiatomaceous earth, and the filter cake was washed with diethyl ether(10 mL) and then ethyl acetate (10 mL). The combined filtrates wereextracted with 6M hydrochloric acid (3×4 mL). The aqueous extracts werecombined and concentrated on a rotary evaporator to yield 1.6 g ofN,3-dimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclopropan]-3-aminehydrochloride as a white solid (53% yield). ¹H NMR (400 MHz, D₂O): δ2.52 (s, 1H), 2.46 (s, 3H), 1.72 (d, J=11 Hz, 1H), 1.49-1.41 (m, 3H),1.39-1.32 (m, 2H), 1.28 (d, J=11 Hz, 1H), 1.02 (s, 3H), 0.59-0.51 (m,3H), 0.45-0.43 (m, 1H); LCMS (m/z): 166 (M+1).

Example 9:N,3-dimethylspiro[bicyclo[2.2.2]oct[5]ene-2,1′-cyclopentan]-3-aminehydrochloride andN,3-dimethylspiro[bicyclo[2.2.2]octane-2,1′-cyclopentan]-3-aminehydrochloride

The intermediate, bicyclo[2,2,2]oct-5-en-2-one, was made usingprocedures described by Kozikowski and Schmiesing, J. Org. Chem. 48:1000-1007 (1983), previously incorporated by reference with regard tosuch reaction, and then subsequently transformed intoN,3-dimethylspiro[bicyclo[2.2.2]oct[5]ene-2,1′-cyclopentan]-3-amine andN,3-dimethylspiro[bicyclo[2.2.2]octane-2,1′-cyclopentan]-3-amine.

A mixture of acrylonitrile (79.4 g, 1.49 mol), 1,3-cyclohexadiene (60 g,0.75 mol), and hydroquinone (1.1 g, 10 mmol) was sealed in a tube andheated at 120° C. for 18 h. The resulting mixture was concentrated andpurified by chromatography on silica gel, eluting with mixtures of ethylacetate (0.5% to 1%) in petroleum ether, to give a separable mixture ofisomers (presumably endo/exo) of 5-cyanobicyclo[2,2,2]oct-2-ene (64 g,64% yield) as a white semisolid. ¹H NMR (300 MHz, CDCl₃) δ 1.32 (m, 2H),1.75 (m, 3H), 2.04 (m, 1H), 2.43 (m, 1H), 2.62 (m, 1H), 2.78 (m, 1H),6.23 (m, 1H), 6.30 (m, 1H); ¹H NMR (300 MHz, CDCl₃) δ 1.28 (m, 2H), 1.50(m, 3H), 1.94 (m, 1H), 2.68 (m, 2H), 2.87 (m, 1H), 6.29 (m, 1H), 6.44(m, 1H); LCMS (m/z), 134 (M+1).

To a refluxing mixture of pyridine (14.2 g, 0.180 mol), phosphoruspentachloride (28.0 g, 0.135 mol), and chloroform (100 mL) was addeddrop-wise a solution of 5-cyanobicyclo[2,2,2]oct-2-ene (12 g, 90 mmol)in chloroform (50 mL). The resulting mixture was heated at reflux for 15h, cooled, and poured onto ice. The organic layer was concentrated, andthe residue was purified by chromatography on silica gel, eluting withmixtures of ethyl acetate (0.5% to 1%) in petroleum ether, to give5-chloro-5-cyanobicyclo[2,2,2]oct-2-ene (14.3 g, 95% yield) as a whitesemisolid. ¹H NMR (300 MHz, CDCl₃) δ 1.3-1.5 (m, 3H), 2.02-2.18 (m, 2H),2.51 (m, 1H), 2.72 (m, 1H), 3.12 (m, 1H), 6.22 (m, 1H), 6.41 (m, 1H);GCMS (m/z): 167.

To a stirred solution of 5-chloro-5-cyanobicyclo[2,2,2]oct-2-ene (65 g,0.39 mol) (representing several runs of the foregoing procedure) indimethyl sulfoxide (500 mL) was added potassium hydroxide (87.4 g, 1.56mol) and water (30 mL). The resulting mixture was stirred at roomtemperature for 15 h, diluted with water (1000 mL) and extracted withether (4×500 mL). The combined ether extracts were washed with brine,dried over anhydrous sodium sulfate, and concentrated. The residue waspurified by chromatography on silica gel, eluting with mixtures ofdiethyl ether (1% to 5%) in petroleum ether, to givebicyclo[2,2,2]oct-5-en-2-one (23.8 g, 50% yield) as a white solid. ¹HNMR (300 MHz, CDCl₃) δ 1.53-1.84 (m, 4H), 2.01-2.02 (m, 2H), 2.96-2.99(m, 1H), 3.11-3.13 (m, 1H), 6.15-6.21 (m, 1H), 6.43-6.48 (m, 1H); GCMS(m/z): 122.

n-Butyllithium (56.5 mL of 1.6 M in hexanes, 90.4 mmol) was added to asolution of diisopropylamine (11.2 mL, 8.05 g, 79.6 mmol) in dry THF(108 mL) at −78° C. The mixture was warmed to 0° C. and stirred for 30min. The solution was again cooled to −78° C., andbicyclo[2,2,2]oct-5-en-2-one (5.00 g, 36.2 mmol) dissolved in THF (10mL) was added. The reaction was stirred for 30 min at −78° C., and thenhexamethylphosphoric triamide (13.9 mL, 14.3 g, 79.6 mmol) followed by1,4-dibromobutane (4.76 mL, 8.59 g, 39.8 mmol) were added. The reactionmixture was warmed to ambient temperature, stirred for 16 h, quenchedwith saturated aqueous ammonium chloride (50 mL), diluted with ether(100 mL), and washed with water (3×50 mL). The organic layer was driedover anhydrous sodium sulfate, filtered, and concentrated. The residuewas purified on a 120 g silica column, eluting with 100% hexanes for 4column volumes, followed by a step gradient to 9:1 hexanes/ethylacetate. Selected fractions were concentrated to yieldspiro[bicyclo[2.2.2]oct[5]ene-2,1′-cyclopentan]-3-one (5.1 g; ˜90% pureby GC/MS) as a clear oil. The material was carried forward withoutfurther purification, by dissolving in dry THF (20 mL) and cooling to−78° C. Methylmagneslum bromide (28.6 mL of 3.0 M in diethyl ether, 85.8mmol) was then added, and the reaction was slowly warmed to ambienttemperature. The reaction was stirred at ambient temperature for 18 hand quenched by careful addition of saturated aqueous ammonium chloride.The reaction was transferred to a separatory funnel, and the aqueouslayer was removed. The organic layer was washed twice with water (10mL), dried over anhydrous sodium sulfate, filtered, and concentrated.The remaining material (colorless oil) was a mixture of3-methylspiro[bicyclo[2.2.2]oct[5]ene-2,1′-cyclopentan]-3-ol (4.9 g) andstarting material.

Without further purification, the sample generated immediately above wascombined with sodium cyanide (1.93 g, 37.8 mmol) in acetic acid (20 mL).This mixture was cooled to 0° C. and stirred at that temperature assulfuric acid (20 mL) was slowly added. The reaction, which turned adeep red color upon complete addition of reagents, was stirred atambient temperature for 18 h. It was then quenched with the addition of100 mL water, made basic (pH 9) by addition of 3 M aqueous sodiumhydroxide, and extracted with dichloromethane (4×50 mL). The combinedorganic layers were dried over anhydrous sodium sulfate and concentratedto obtain an off-white solid. The solid was dissolved in dry THF (200mL), cooled to 0° C. and held at this temperature as a solution oflithium aluminum hydride (25.2 mL of 2 M in THF, 50.4 mmol) was slowlyadded. The reaction was heated to reflux for 18 h, cooled in ice-bath,and quenched by cautious addition of 5 g of sodium sulfate decahydrate.The resulting mixture was stirred for 30 min and filtered. The filtratewas concentrated, and the residue was purified on 120 g silica gelcolumn, using 0-70% CMA in chloroform, providingN,3-dimethylspiro[bicyclo[2.2.2]oct[5]ene-2,1′-cyclopentan]-3-amine(0.20 g, 2.7% yield). This material was taken up in dichloromethane (5ml), converted to the HCL salt by treating with 0.5 mL of 4 Mhydrochloric acid in dioxane and concentrating the resulting mixture.The resultant amorphous solid was dissolved in methanol (3 mL) andprecipitated with diethyl ether (3 mL). The solvent was removed byaspiration, and the precipitate was triturated three times with diethylether (3 mL). The sample of hydrochloride salt was then vacuum dried. ¹HNMR (300 MHz, CD₃OD) δ 1.02 (s, 3H), 1.19-1.42 (m, 4H), 1.51-1.64 (m,7H), 1.81 (m, 1H), 2.21 (m, 2H), 2.73 (s, 3H), 5.57 (dd, J₁=9 Hz, J₂=3Hz, 1H), 6.01 (d, J=6 Hz, 1H); LCMS (m/z): 206 (M+1).

N,3-dimethylspiro[bicyclo[2.2.2]oct[5]ene-2,1′-cyclopentan]-3-amine (80mg, 0.39 mmol) was dissolved in methanol (7.8 mL) and 10% Pd/C (wet) (41mg) was added. This mixture was placed under a balloon of hydrogen gasand stirred at ambient temperature for 16 h. The reaction mixture wasthen filtered through diatomaceous earth, and the filtrate wasconcentrated, leavingN,3-dimethylspiro[bicyclo[2.2.2]octane-2,1′-cyclopentan]-3-amine (45 mg,56% yield). This was dissolved in dichloromethane (3 mL), converted toits hydrochloric acid salt by treating with 0.3 mL of 4 M hydrochloricacid in dioxane, and concentrating the resulting mixture. The resultantamorphous solid was dissolved in methanol (3 mL) and precipitated withdiethyl ether (1 mL). The solvent was removed by aspiration, and theprecipitate was triturated three times with diethyl ether (3 mL). Thesample of hydrochloride salt was then vacuum dried. ¹H NMR (300 MHz,CD₃OD) δ 0.99 (s, 3H), 1.31 (m, 2H), 1.45-1.70 (m, 10H), 1.85 (m, 1H),2.08 (m, 3H), 2.22-2.35 (m, 1H), 2.65 (s, 3H), 3.02 (m, 1H); LCMS (m/z):208 (M+1).

VI. Biological Assays Characterization of Interactions at NicotinicAcetylcholine Receptors Materials and Methods

Cell Lines.

SH-EP1-human α4β2 (Eaton et al., 2003), SH-EP1-human α4β4 (Gentry etal., 2003) and SH-EP1-α6β3β4α5 (Grinevich at al., 2005) cell lines wereobtained from Dr. Ron Lukas (Barrow Neurological Institute). The SH-EP1cell lines, PC12, SH-SY5Y and TE671/RD cells were maintained inproliferative growth phase in Dulbecco's modified Eagle's medium(Invitrogen, Carlsbad, Calif.) with 10% horse serum (Invitrogen), 5%fetal bovine serum (HyClone, Logan Utah), 1 mM sodium pyruvate, 4 mML-glutamine. For maintenance of stable transfectants, the α4β2 and α4β4cell media was supplemented with 0.25 mg/mL zeocin and 0.13 mg/mLhygromycin B. Selection was maintained for the α6β3β4α5 cells with 0.25mg/mL of zeocin, 0.13 mg/mL of hygromycin B, 0.4 mg/mL of geneticin, and0.2 mg/mL of blasticidin.

Receptor Binding Assays

Preparation of Membranes from Rat Tissues.

Rat cortices were obtained from Analytical Biological Services,Incorporated (ABS, Wilmington, Del.). Tissues were dissected from femaleSprague-Dawley rats, frozen and shipped on dry ice. Tissues were storedat −20° C. until needed for membrane preparation. Cortices from 10 ratswere pooled and homogenized by Polytron (Kinematica GmbH, Switzerland)in 10 volumes (weight:volume) of ice-cold preparative buffer (11 mM KCl,6 mM KH₂PO₄, 137 mM NaCl, 8 mM Na₂HPO₄, 20 mM HEPES (free acid), 5 mMiodoacetamide, 1.5 mM EDTA, 0.1 mM PMSF pH 7.4). The resultinghomogenate was centrifuged at 40,000 g for 20 minutes at 4° C. and theresulting pellet was re-suspended in 20 volumes of ice-cold water. After60 minute Incubation at 4° C., a new pellet was collected bycentrifugation at 40,000 g for 20 minutes at 4° C. The final pellet wasre-suspended in preparative buffer and stored at −20° C. On the day ofthe assay, tissue was thawed, centrifuged at 40,000 g for 20 minutes andthen re-suspended in Dulbecco's Phosphate Buffered Saline, pH 7.4 (PBS,Invitrogen) to a final concentration of 2-3 mg protein/mL. Proteinconcentrations were determined using the Pierce BCA Protein Assay kit(Pierce Biotechnology, Rockford, Ill.), with bovine serum albumin as thestandard.

Preparation of Membranes from Clonal Cell Lines.

Cells were harvested in ice-cold PBS, pH 7.4, then homogenized with aPolytron (Kinematica GmbH, Switzerland). Homogenates were centrifuged at40,000 g for 20 minutes (4° C.). The pellet was re-suspended in PBS andprotein concentration determined using the Pierce BCA Protein Assay kit(Pierce Biotechnology, Rockford, Ill.).

Competition Binding to Receptors in Membrane Preparations.

Binding to nicotinic receptors was assayed on membranes using standardmethods adapted from published procedures (Lippiello and Fernandes 1986;Davies et al., 1999). In brief, membranes were reconstituted from frozenstocks and incubated for 2 h on ice in 150 μl assay buffer (PBS) in thepresence of competitor compound (0.001 nM to 100 μM) and radioligand.[³H]-nicotine (L-(−)-[N-methyl-³H]-nicotine, 69.5 Ci/mmol, Perkin-ElmerLife Sciences, Waltham, Mass.) was used for human α4β2 binding studies.[³H]-epibatidine (52 Ci/mmol, Perkin-Elmer Life Sciences) was used forbinding studies at the other nicotinic receptor subtypes.L-[Benzilic-4,4-³H] Quinuclidinyl Benzilate ([³H]QNB) was used formuscarinic receptor binding studies. Incubation was terminated by rapidfiltration on a multimanifold tissue harvester (Brandel, Gaithersburg,Md.) using GF/B filters presoaked in 0.33% polyethyleneimine (w/v) toreduce non-specific binding. Filters were washed 3 times with ice-coldPBS and the retained radioactivity was determined by liquidscintillation counting.

Binding Data Analysis.

Binding data were expressed as percent total control binding. Replicatesfor each point were averaged and plotted against the log of drugconcentration. The IC₅₀ (concentration of the compound that produces 50%inhibition of binding) was determined by least squares non-linearregression using GraphPad Prism software (GraphPAD, San Diego, Calif.).Ki was calculated using the Cheng-Prusoff equation (Cheng and Prusoff,1973).

Calcium Flux Functional Assays

Twenty-four to forty-eight hours prior to each experiment, cells wereplated in 96 well black-walled, clear bottom plates (Corning, Corning,N.Y.) at 60-100,000 cells/well. On the day of the experiment, growthmedium was gently removed, 200 μL 1×FLIPR Calcium 4 Assay reagent(Molecular Devices, Sunnyvale, Calif.) in assay buffer (20 mM HEPES, 7mM TRIS base, 4 mM CaCl₂, 5 mM D-glucose, 0.8 mM MgSO₄, 5 mM KCl, 0.8 mMMgCl₂, 120 mM N-methyl D-glucamine, 20 mM NaCl, pH 7.4 for SH-EP1-humanα4β2 cells or 10 mM HEPES, 2.5 mM CaCl₂, 5.6 mM D-glucose, 0.8 mM MgSO₄,5.3 mM KCl, 138 mM NaCl, pH 7.4 with TRIS-base for all other cell lines)was added to each well and plates were incubated at 37° C. for 1 hour(29° C. for the 29° C.-treated SH-EP1-human α4β2 cells). For inhibitionstudies, competitor compound (10 pM-10 μM) was added at the time of dyeaddition. The plates were removed from the incubator and allowed toequilibrate to room temperature. Plates were transferred to a FLIPRTetra fluorometric Imaging plate reader (Molecular Devices) for additionof compound and monitoring of fluorescence (excitation 485 nm, emission525 nm). The amount of calcium flux was compared to both a positive(nicotine) and negative control (buffer alone). The positive control wasdefined as 100% response and the results of the test compounds wereexpressed as a percentage of the positive control. For inhibitionstudies, the agonist nicotine was used at concentrations of 1 μM forSH-EP1-human α4β2 and SH-EP1-human α4β4 cells, 10 μM for PC12 andSH-SY5Y cells, and 100 μM for TE671/RD cells.

Neurotransmitter Release

Dopamine release studies were performed using striatal synaptosomesobtained from rat brain as previously described (Bencherif et al.,1998). Striatal tissue from two rats (female, Sprague-Dawley, weighing150-250 g) was pooled and homogenized in ice-cold 0.32 M sucrose (8 mL)containing 5 mM HEPES, pH 7.4, using a glass/glass homogenizer. Thetissue was then centrifuged at 1,000×g for 10 minutes. The pellet wasdiscarded and the supernatant was centrifuged at 12,500×g for 20minutes. The resulting pellet was re-suspended in ice-cold perfusionbuffer containing monoamine oxidase inhibitors (128 mM NaCl, 1.2 mMKH₂PO₄, 2.4 mM KCl, 3.2 mM CaCl₂, 1.2 mM MgSO₄, 25 mM HEPES, 1 mMascorbic acid, 0.02 mM pargyline HCl and 10 mM glucose, pH 7.4) andcentrifuged for 15 minutes at 23,000×g. The final pellet wasre-suspended in perfusion buffer (2 mL) for immediate use.

The synaptosomal suspension was incubated for 10 minutes in a 37° C.shaking incubator to restore metabolic activity. [³H]Dopamine ([³H]DA,specific activity=28.0 Ci/mmol, NEN Research Products) was added at afinal concentration of 0.1 μM and the suspension was incubated at 37′Cfor another 10 minutes. Aliquots of perfusion buffer (100 μL) and tissue(100 μL) were loaded into the suprafusion chambers of a BrandelSuprafusion System (series 2500, Gaithersburg, Md.). Perfusion buffer(room temperature) was pumped into the chambers at a rate ofapproximately 0.6 mL/min for a wash period of 8 min. Competitor compound(10 pM-100 nM) was applied in the perfusion stream for 8 minutes.Nicotine (10 μM) was then applied in the perfusion stream for 48seconds. Fractions (12 seconds each) were continuously collected fromeach chamber throughout the experiment to capture basal release andagonist-induced peak release and to re-establish the baseline after theagonist application. The perfusate was collected directly intoscintillation vials, to which scintillation fluid was added. Released[³H]DA was quantified by scintillation counting. For each chamber, theintegrated area of the peak was normalized to its baseline.

Release was expressed as a percentage of release obtained with controlnicotine in the absence of competitor. Within each assay, each testcompound concentration was replicated using 2 chambers; replicates wereaveraged. The compound concentration resulting in half maximalinhibition (IC₅₀) of specific ion flux was defined.

Patch Clamp Electrophysiology

Cell Handling.

After removal of GH4C1-rat T6′S α7 cells from the incubator, medium wasaspirated, cells trypsinized for 3 minutes, gently triturated to detachthem from the plate, washed twice with recording medium, andre-suspended in 2 ml of external solution (see below for composition).Cells were placed in the Dynaflow chip mount on the stage of an invertedZeiss microscope (Carl Zeiss Inc., Thornwood, N.Y.). On average, 5minutes was necessary before the whole-cell recording configuration wasestablished. To avoid modification of the cell conditions, a single cellwas recorded per single load. To evoke short responses, compounds wereapplied for 0.5 s using a Dynaflow system (Cellectricon, Inc.,Gaithersburg, Md.), where each channel delivered pressure-drivensolutions at either 50 or 150 psi.

Electrophysiology.

Conventional whole-cell current recordings were used. Glassmicroelectrodes (5-10 MΩ resistance) were used to form tight seals (>1GΩ) on the cell surface until suction was applied to convert toconventional whole-cell recording. The cells were then voltage-clampedat holding potentials of −60 mV, and ion currents in response toapplication of ligands were measured. Whole-cell currents recorded withan Axon 700A amplifier were filtered at 1 kHz and sampled at 5 kHz by anADC board 1440 (Molecular Devices). Whole-cell access resistance wasless than 20 MΩ. Data acquisition of whole-cell currents was done usinga Clampex 10 (Molecular Devices, Sunnyvale, Calif.), and the resultswere plotted using Prism 5.0 (GraphPad Software Inc., San Diego,Calif.). The experimental data are presented as the mean±S.E.M., andcomparisons of different conditions were analyzed for statisticalsignificance using Student's t and Two Way ANOVA tests. All experimentswere performed at room temperature (22±1° C.). Concentration-responseprofiles were fit to the Hill equation and analyzed using Prism 5.0.

Solutions and Drug Application.

The standard external solution contained: 120 mM NaCl, 3 mM KCl, 2 mMMgCl₂, 2 mM CaCl₂, 25 mM D-glucose, and 10 mM HEPES and was adjusted topH 7.4 with Tris base. Internal solution for whole-cell recordingsconsisted of: 110 mM Tris phosphate dibasic, 28 mM Tris base, 11 mMEGTA, 2 mM MgCl₂, 0.1 mM CaCl₂, and 4 mM Mg-ATP, pH 7.3. (Liu et al.,2008). To initiate whole-cell current responses, compounds weredelivered by moving cells from the control solution toagonist-containing solution and back so that solution exchange occurredwithin ˜50 ms (based on 10-90% peak current rise times). Intervalsbetween compound applications (0.5-1 min) were adjusted specifically toensure the stability of receptor responsiveness (without functionalrundown), and the selection of pipette solutions used in most of thestudies described here was made with the same objective. (−)-Nicotineand acetylcholine (ACh), were purchased from Sigma-Aldrich (St. Louis,Mo.). All drugs were prepared daily from stock solutions.

To determine the inhibition of ACh induced currents by compounds of thepresent invention, we established a stable baseline recording applying70 μM ACh (usually stable 5-10 consecutive applications). Then ACh (70μM) was co-applied with test compound in a concentration range of 1 nMto 10 μM. Since tall of the current (current measured at the end of 0.5s ACh application) underwent the most profound changes, inhibition andrecovery plots represent amplitude of tall current.

Tabulated Summary

As shown in Table 1, compounds representative of the present inventiontypically exhibit inhibition constants (Ki values) for human α4β2 andganglionic receptor subtypes in the 1-100 mM range, indicating a lowaffinity for the orthosteric binding sites (i.e. the binding site of thecompetitive agonist) of these receptor subtypes. The data in Table 1,however, also illustrates that compounds representative of the presentinvention effectively Inhibit ion flux for these receptor subtypes, withtypical IC₅₀ values of less than about 2 mM and typical I_(max) valuesof >95%. Taken together, this data demonstrates that the compoundsrepresentative of this invention are effective at inhibiting ion fluxmediated by these receptor subtypes through a mechanism that does notinvolve binding at the orthosteric sites.

TABLE 4 Human Human Human Human α4β2 Ca α4β2 Ca α4β2 Ca α4β2 Ca HumanHuman Human Human Flux IC50 Flux Imax Flux IC50 Flux Imax Ganglion CaGanglion Ca α4β2 Ki Ganglion Ki [29C/HS] [29C/HS] [37C/LS] [37C/LS] FluxIC50 Flux Imax Structure (nM) (nM) (nM) (% inh) (nM) (% inh) (nM) (%inh)

>10,000 >10,000 1000 99 500 97 160 97

3800 >10,000 1400 99 59 96 190 79

10,000 >10,000 1400 96 740 98 190 89

2600 >10,000 1800 98 620 98 230 98

540 >10,000 2100 97 570 93 440 93

8700 >10,000 610 98 230 95 98 93

>10,000 >10,000 210 98 97 96 23 100

>10,000 >10,000 260 98 65 94 40 98

>10,000 >10,000 1300 99 490 96 180 97

5600 >10,000 710 98 430 96 74 99

>10,000 >10,000 1000 97 600 96 98 95

>10,000 >10,000 310 97 270 93 60 97

>10,000 >10,000 540 96 520 97 100 98

>10,000 >10,000 140 96 350 95 66 97

3200 >10,000 1100 96 280 95 65 95

2600 >10,000 740 95 380 95 86 99

>10,000 >10,000 1100 99 120 100 35 96

>10,000 >10,000 380 97 120 93 170 98

3000 >10,000 400 95 240 92

The specific pharmacological responses observed may vary according toand depending on the particular active compound selected or whetherthere are present pharmaceutical carriers, as well as the type offormulation and mode of administration employed, and such expectedvariations or differences in the results are contemplated in accordancewith practice of the present invention.

Although specific embodiments of the present invention are hereinillustrated and described in detail, the invention is not limitedthereto. The above detailed descriptions are provided as exemplary ofthe present invention and should not be construed as constituting anylimitation of the invention. Modifications will be obvious to thoseskilled in the art, and all modifications that do not depart from thespirit of the invention are intended to be Included with the scope ofthe appended claims.

What is claimed is: 1.-15. (canceled)
 16. A compoundexo-N,3-dimethylspiro[bicycle[2.2.1]heptane-2,1′-cyclobutan]-3-amine ora pharmaceutically acceptable salt thereof.
 17. A compound

(1S,3S,4R)—N,3-dimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-amineor a pharmaceutically acceptable salt thereof.
 18. A compound

(1R,3R,4S)—N,3-dimethylspiro[bicyclo[2.2.1]heptane-2,1′-cyclobutan]-3-amineor a pharmaceutically acceptable salt thereof.